Cell-cell cohesion is a defining feature of multicellular organisms, and a number of disease states, from cancer to heart disease, are driven by defects in the structural Velcro that holds cells to one another. It is widely viewed that the cadherin/catenin adhesion complex is a master regulator of this adhesive Velcro. This complex comprises a transmembrane cadherin component that mediates Ca++-dependent homophilic recognition, and a number of associated catenins that link cadherins to the underlying cytoskeleton. Since alpha-catenin (?-cat) is the sole actin-binding component of the cadherin/catenin complex, and filamentous (F)- actin is critical for strong intercellular adhesion, ?-cat has long been viewed as a key linker protein between cadherin adhesion receptors and the actin cytoskeleton. While loss of function studies show that ?-cat is essential for cell-cell adhesion across many cell types, the means through which this occurs is poorly understood. Our lab has discovered a highly conserved phosphorylation domain in ?-cat that is situated just N- terminal to a filamentous actin-binding site. These phosphorylations also lie within a mechano-sensitive, auto-inhibitory region of ?-cat, which restricts recruitment of another actin-binding protein to the cadherin/catenin complex via the central region of ?-cat. This proposal, therefore, seeks to understand how phosphorylation directs the conformational regulation, actin-binding and dynamic cell-cell adhesive functions of ?-cat using both in vitro and cell-based assays. Towards this end, Aim 1 seeks to show how in vitro phosphorylation of ?-cat at S641 by casein kinase 2 (CK2) enhances or primes for phosphorylation by CK1 at residues S652, S655 and T658, resulting in a more open conformation that favors ?-cat binding to F-actin and other actin-binding proteins. Aim 2 will determine the contribution of these phosphorylations to cell adhesion in vivo, using a dog epithelial cell line that has replaced the endogenous ?-cat with GFP-tagged forms that contain, lack or constitutively mimic phosphorylation. Together, these aims address the fundamental question of how ?-cat interacts with actin at specialized adhesive junctions using both in vitro and cell-based approaches, which will have broad implications for intercellular adhesion across diverse cell types, and may be relevant to pathophysiological conditions that impact epithelial barrier function and overall tissue integrity.